Abstract: :
Purpose: Monitoring of transgene expression in live animals,which is facilitated by GFPs, is desirable for glaucoma genetherapy experimentation. In explanted human eyes, expressionof eGFP or of ß-galactosidase (ß-gal) with FIV vectorsis associated with minor and transient outflow facility changesdespite excellent gene transfer and marker protein over-expressionin TM. Here we assessed 3 different marker transgenes in vivo.Methods: Nine domestic cats received anterior chamber (AC)bolus injections of transducing unit (TU)-normalized, VSV-Gpseudotyped FIV vectors CT26 (ß-gal), GINWF (eGFP fromAquorea victoria) or RGWF (GFP from Renilla reniformis). Twowere injected with 10^8 TU GINWF into the right eye (R), whilethe left (L) served as an uninjected control. Four receivedGINWF (R) and CT26 (R) (10^8 and 10^7 TU in two cats each).Three additional cats were injected with 10^8 TU RGWF (R) and10^8 TU GINWF (L).Results: Expression plateaued at 17±9days and was confined to the TM except for a few cells in theiris epithelium. High expression was observed for more than52 days with Aequoria eGFP, which produced brilliant fluorescencethroughout the TM but also resulted in subsequent iritis anda 5.7±4.3 mmHg IOP decrease, followed by terminationof eGFP expression several days later. In contrast, loss ofexpression was not seen with ß-gal expression, which persistedand was highly expressed throughout the TM at sacrifice. Systemicand local corticosteroids did not prevent GFP-induced iritis.Iritis was clinically minimal with Renilla GFP, although expressionwas less. Histologically, Aequoria eGFP- and, to a lesser extent,Renilla GFP-transduced TMs, had lymphocytic infiltrates, whileß-gal-transduced TMs did not. In vitro, sera from injectedanimals had low to moderate neutralizing activity (1:100-1:1000dilutions) against both VSV-G- and control lymphocytic choriomeningitisvirus (LCMV) envelope-pseudotyped FIV (and HIV-1) vectors, buteGFP- or VSV-G-specific antibodies were not detected.Conclusions:Aequoria eGFP and Renilla GFP induced clinically and histologicallyevident AC inflammatory responses that were associated withtermination of expression. In contrast, ß-gal did not.Since these initial studies revealing marker protein-specifictoxicity involved maximum over-expression in highly transducedTMs, dose response studies are needed to understand the implications.